Functional Rescue of Retinal Degeneration-Associated Mutant RPE65 Proteins

Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 854)


More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells. The 26S proteasome non-ATPase regulatory subunit 13, a newly identified negative regulator of RPE65, mediated degradation of mutant RPE65s, which were misfolded and formed aggregates in the cells. Many mutations, including L22P, T101I, and L408P, were mapped on nonactive sites of RPE65. Enzyme activities of these mutant RPE65s were significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. 4-phenylbutyrate (PBA) displayed a significant synergistic effect on the low temperature-mediated rescue of the mutant RPE65s. Our results suggest that a low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy for delaying retinal degeneration caused by RPE65 mutations.


RPE65 Retinoid Visual cycle Leber congenital amaurosis Retinitis pigmentosa PSMD13 Proteasome Low temperature Chemical chaperone Gene therapy Retina 



This work was supported by NIH grants (EY021208 to M. J., EY017280 to S. G. J., and GM103340 to LSU Neuroscience COBRE facility), Macula Vision Research Foundation grants (to D. B. and to S.G.J.), and a Research to Prevent Blindness grant (to LSU Ophthalmology).


  1. Bonapace G, Waheed A, Shah GN et al (2004) Chemical chaperones protect from effects of apoptosis-inducing mutation in carbonic anhydrase IV identified in retinitis pigmentosa 17. Proc Natl Acad Sci U S A 101:12300–12305CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bowne SJ, Humphries MM, Sullivan LS et al (2011) A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement. Eur J Hum Genet 19:1074–1081CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chen Y, Moiseyev G, Takahashi Y et al (2006) Impacts of two point mutations of RPE65 from Leber’s congenital amaurosis on the stability, subcellular localization and isomerohydrolase activity of RPE65. FEBS Lett 580:4200–4204CrossRefPubMedGoogle Scholar
  4. Cideciyan AV, Aleman TS, Boye SL et al (2008) Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci U S A 105:15112–15117CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cideciyan AV, Jacobson SG, Beltran WA et al (2013) Human retinal gene therapy for Leber congenital amaurosis shows advancing retinal degeneration despite enduring visual improvement. Proc Natl Acad Sci U S A 110:E517–E525CrossRefPubMedPubMedCentralGoogle Scholar
  6. Denning GM, Anderson MP, Amara JF et al (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358:761–764CrossRefPubMedGoogle Scholar
  7. Dunn KC, Aotaki-Keen AE, Putkey FR et al (1996) ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62:155–169CrossRefPubMedGoogle Scholar
  8. Hauswirth WW, Aleman TS, Kaushal S et al (2008) Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther 19:979–990CrossRefPubMedPubMedCentralGoogle Scholar
  9. Hu J, Bok D (2001) A cell culture medium that supports the differentiation of human retinal pigment epithelium into functionally polarized monolayers. Mol Vis 7:14–19PubMedGoogle Scholar
  10. Jin M, Li S, Moghrabi WN et al (2005) Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium. Cell 122:449–459CrossRefPubMedPubMedCentralGoogle Scholar
  11. Jin M, Yuan Q, Li S et al (2007) Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65. J Biol Chem 282:20915–20924CrossRefPubMedPubMedCentralGoogle Scholar
  12. Kiser PD, Golczak M, Lodowski DT et al (2009) Crystal structure of native RPE65, the retinoid isomerase of the visual cycle. Proc Natl Acad Sci U S A 106:17325–17330CrossRefPubMedPubMedCentralGoogle Scholar
  13. Li S, Yang Z, Hu J et al (2013a) Secretory defect and cytotoxicity: the potential disease mechanisms for the retinitis pigmentosa (RP)-associated interphotoreceptor retinoid-binding protein (IRBP). J Biol Chem 288:11395–11406CrossRefPubMedPubMedCentralGoogle Scholar
  14. Li S, Lee J, Zhou Y et al (2013b) Fatty acid transport protein 4 (FATP4) prevents light-induced degeneration of cone and rod photoreceptors by inhibiting RPE65 isomerase. J Neurosci 33:3178–3189CrossRefPubMedPubMedCentralGoogle Scholar
  15. Li S, Izumi T, Hu J et al (2014) Rescue of enzymatic function for disease-associated RPE65 proteins containing various missense mutations in non-active sites. J Biol Chem 289:18943–18956CrossRefPubMedPubMedCentralGoogle Scholar
  16. Maguire AM, Simonelli F, Pierce EA et al (2008) Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med 358:2240–2248CrossRefPubMedPubMedCentralGoogle Scholar
  17. Moiseyev G, Chen Y, Takahashi Y et al (2005) RPE65 is the isomerohydrolase in the retinoid visual cycle. Proc Natl Acad Sci U S A 106:12413–12418CrossRefGoogle Scholar
  18. Nikolaeva O, Takahashi Y, Moiseyev G et al (2010) Negative charge of the glutamic acid 417 residue is crucial for isomerohydrolase activity of RPE65. Biochem Biophys Res Commun 391:1757–1761CrossRefPubMedPubMedCentralGoogle Scholar
  19. Philp AR, Jin M, Li S et al (2009) Predicting the pathogenicity of RPE65 mutations. Hum Mutat 30:1183–1188CrossRefPubMedPubMedCentralGoogle Scholar
  20. Redmond TM, Poliakov E, Yu S et al (2005) Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci U S A 102:13658–13663CrossRefPubMedPubMedCentralGoogle Scholar
  21. Rubenstein RC, Zeitlin PL (1998) Use of protein repair therapy in the treatment of cystic fibrosis. Curr Opin Pediatr 10:250–255CrossRefPubMedGoogle Scholar
  22. Sato K, Li S, Gordon WC et al (2013) Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein. J Neurosci 33:17458–17468CrossRefPubMedPubMedCentralGoogle Scholar
  23. Takahashi Y, Chen Y, Moiseyev G et al (2006) Two point mutations of RPE65 from patients with retinal dystrophies decrease the stability of RPE65 protein and abolish its isomerohydrolase activity. J Biol Chem 281:21820–21826CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Department of Ophthalmology and Neuroscience CenterLouisiana State University Health Sciences CenterNew OrleansUSA
  2. 2.Jules Stein Eye InstituteUniversity of CaliforniaLos AngelesUSA
  3. 3.Department of BiologyWashington UniversitySt. LouisUSA
  4. 4.Scheie Eye Institute, Department of OphthalmologyPerelman School of Medicine, University of PennsylvaniaPhiladelphiaUSA

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